Salmonella enterica serotype Heidelberg (S. Heidelberg) is a group B
Salmonella which apparently accounts for a small proportion of cases
of disease in humans. Between 1994 and 1997, S. Heidelberg was the tenth
most frequently identified serotype isolated from humans in Italy (1.3%
of all human isolates), although it does not appear among the top ten
serotypes from the data from the Enter-Net Italia surveillance system
available for 1999, 2000 and 2001. National data from the veterinary
surveillance system of Salmonella serotypes do however confirm the presence
of S. Heidelberg in the poultry farm environment, this serotype being
identified in 2002 from 6.5% and 20.3%, respectively, of Salmonella
strains from chicken and turkey (1).
Reports from countries including the United States (US) and Canada describe
a high prevalence of the Heidelberg serotype in both human and non-human
sources, mainly food and livestock (2-4). A nursing home outbreak attributable
to this serotype, associated with Campylobacter jejuni, has also been
reported by Layton et al (5).
In January 2002, a small family cluster of infections of S. Heidelberg,
involving two children and their asymptomatic father, who was working
in a poultry slaughterhouse, occurred in Pistoia, Tuscany. The strain
exhibited plasmid-mediated resistances to ampicillin and sulphonamides.
At the same time, in Palermo, Sicily, one S. Heidelberg strain with
identical drug susceptibility pattern and plasmid profile was isolated
from retail commercial chicken entrails. Between March and June 2003,
S. Heidelberg was again identified at the Centro per gli Enterobatteri
Patogeni per l'Italia Meridionale (Centre for enteric pathogens for
southern Italy, CEPIM), from two paediatric cases and two samples of
whole chicken on sale.
A typing study by phenotypic and molecular techniques was thus performed
to substantiate a possible relationship between S. Heidelberg isolates
from these two areas of Italy. A retrospective analysis of available
isolates identified in the years 1999-2001 from different regions of
Italy was also carried out.
Materials and methods
The study included 21 isolates from human cases, chicken products and
the environment from different regions of Italy (Table). Epidemiological
data about possible exposures to contaminated sources (eating of particular
foods, previous travel or farm visit) were unavailable for most cases.
All samples of chicken were on sale in Palermo and Agrigento (Sicily),
but processed and marketed by two poultry factories with nationwide
Susceptibility patterns were assessed by disk diffusion according to
the criteria of the National Committee for Clinical Laboratory Standards
(6). Plasmid content was investigated by the alkaline lysis method (7).
PFGE was performed by a standard procedure (8). The DNA size standard
used was the bacteriophage lambda ladder ranging from 48.5 to 1.000
kb (Bio-Rad). Macrorestriction fragment patterns were visually
analyzed and classified by previously established guidelines (9, 10).
Isolates with electrophoretic patterns differing by one to four DNA
fragments were classified as subtypes of the same pulsetype. For the
purpose of this study, different pulsotypes were considered to identify
distinct clones of the Heidelberg serotype.
Five distinctive antimicrobial resistance profiles were identified among
the S. Heidelberg isolates under study (Table 1). The predominant pattern
- 11 of 21 isolates - was characterized by resistance to ampicillin,
streptomycin, tetracycline and nalidixic acid, with the addition of
kanamycin in two cases. Since 2001 the ampicillin-sulfonamides resistance
pattern became more frequent. Two strains only were susceptible to all
the antimicrobial agents tested.
Six plasmid profiles - a to f - were identified among 19 of the 21 isolates
(Table 1 and Fig. 1). The two fully susceptible isolates were also plasmid
Three distinct XbaI PFGE patterns, designated X-A to X-C were observed
(Fig. 2). Within PFGE type X-A pattern there were three subtypes X-A1,
X-A2 and X-A3. PFGE subtype X-A1 accounted for 16 of 21 isolates, whilst
subtypes X-A2 and X-A3 were represented by a single isolate, respectively
(Table 1). The DNAs of the 16 strains with XbaI-PFGE pattern X-A1 were
digested by BlnI. Fourteen were indistinguishable and were assigned
the pattern B-A1; the remaining two were assigned two different subtypes
B-A2 and B-A3, differing from B-A1 by two bands each (data not shown).
The strains of S. Heidelberg investigated in this study cannot be considered
as a representative sample of the strains circulating in Italy because
of the selection method used. This is an inherent limit of passive surveillance
systems, such as those set up in most European countries for enteric
pathogens, that depend on voluntary adherence by peripheral laboratories.
Nevertheless, these surveillance networks are often able to trace the
transmission routes of some Salmonella clones and describe time and
space trends of the serotypes of major interest to Public Health.
PFGE patterns appear to be consistent with the dissemination of a common
outbreak strain, though different drug susceptibility and plasmid profiles
have been found. PFGE analysis has indeed proved to be a very effective
tool in determining whether some isolates are essentially clonal, and
its application as the basic method in molecular surveillance networks
of verocytotoxigenic Escherichia coli and Salmonella has already been
successful for some years (11). Furthermore, the results of a recent
study on epidemiology of S. Heidelberg in the US have demonstrated the
presence of a clonal strain over a ten year period, thus suggesting
the possible persistence of a particular strain over a wide area for
a prolonged period (12). Moreover, the results obtained concur with
reports by other authors who have used PFGE to identify clonal relationship
between Salmonella isolates, but found different antimicrobial resistance
patterns and plasmid profiles within an apparently unique chromosomal
clone (6,13,14). In our case too, the intrinsic instability of extra-chromosomal
DNA, the selective pressure by use of different antibiotics in different
places, and the broad period of observation might justify the heterogeneity
of the Heidelberg isolates on the basis of their plasmid and drug resistance
patterns. On the other hand, more strain-specific markers, such as the
drug resistance pattern/plasmid profile, might be more successful than
PFGE alone in delineating local transmission routes triggered by clonally
spreading Salmonella serotypes, such as Enteritidis and, presumably,
The association of such a serotype with avian hosts and eggs has been
known for some time. S. Heidelberg was the predominant serotype recovered
from ovary samples in a survey of layer flocks in 1991, and its ability
to penetrate and grow into the interior of hens' eggs has been well
documented (3,15). Moreover, the high prevalence of nalidixic acid resistance
within strains of S. Heidelberg, a zoonotic serotype closely associated
with chickens and turkeys, could be related to their ecological niche.
Indeed, the use of fluoroquinolones is common in the poultry industry
and is temporally strongly associated with raising frequencies of resistances
in many Salmonella serotypes, including Enteritidis (16,17).
Clonal diffusion of some predominant genotypes may be more widespread
than is currently recognized and involve Salmonella strains other than
the traditional Enteritidis and Typhimurium DT104 complex. In Italy,
Heidelberg serotype is apparently able to spread clonally nationwide
through the poultry vehicle. A larger study involving a more representative
sample of strains from Italy and, possibly, other European countries
might more confidently evaluate epidemiological features of this Salmonella
serotype. Molecular epidemiological monitoring should be a routine tool
for detection, and quantitative assessment of unexpected events related
to zoonotic serotypes.